Most of the cortisol in circulating blood is bound to proteins (transcortin and albumin), and the rest is protein-unbound. It has generally been accepted that transcortin-bound cortisol (Tr-F) and albumin-bound cortisol (Al-F) are physiologically inactive, and that protein-unbound cortisol (U-F) is active. It is also known that the binding capacity of transcortin to cortisol is lower at 37 degrees C than at 4 degrees C. However, no investigations concerning the influence of temperature changes the physiological range have been reported, probably because of the lack of proper methods. In this report, the influence of temperature changes the physiological range on serum protein-cortisol binding is assessed and its physiological roles are discussed. Cortisol binding of transcortin and albumin was determined by a newly developed isocolloidosmolar equilibrium dialysis method. The total cortisol concentration was determined by the CPB assay method described by Murphy. In the beginning, experiments were performed using pooled human serum. This serum was dialyzed at 35, 37, 39 and 41 degrees C. U-F and Al-F concentrations increased in accordance with the increment of incubation temperature, whereas Tr-F concentration decreased. The association constant of transcortin to cortisol decreased with the rise of incubation temperature. It was 3.43, 2.92, 2,32 and 1.66 X 10(7)M-1 at 35, 37, 39 and 41 degrees C, respectively. However, the association constant of albumin to cortisol was 3.04 X 10(3)M-1 and stayed unchanged under these conditions. These results suggest that the transcortin-cortisol complex dissociates with temperature rises and that a part of freed cortisol is bound to albumin while the other part remains protein-unbound cortisol. When various amounts of cortisol were added to the pooled human serum, which was dialyzed at 35, 37, 39 and 41 degrees C; U-F, Al-F and Tr-F concentrations increased corresponding to the amount of added cortisol. The increase of U-F, Al-F concentrations and decrease of Tr-F concentration induced by the rise of temperature was still significant at each total coritsol concentration. Cortisol fractions in the serum of healthy subjects were compared with those of 39 febrile patients consisting of A group (body temperature 37 approximately 38 degrees C), B group (38 approximately 39 degrees C), and C group (39 approximately 40 degrees C). Total cortisol concentrations increased with the rise of body temperature. They were 7.62 (normal), 10.64 (A), 14.84 (B) and 21.37 (C) microgram/100ml, respectively. U-F% and Al-F% increased parallel with the total cortisol concentration. Tr-F%, binding capacity of transcortin. These results reveal that the rise of body temperature elevates the total cortisol concentration, and the greater part of increased cortisol becomes U-F and Al-F, while a relatively small part of it is bound to transcortin. The percent change of the total cortisol concentration in comparison with healthy subjects was 150 (A), 209 (B) and 593 (C), respectively...